Chloroperoxidase-catalyzed degradation of pharmaceutical pollutants in wastewater

ABSTRACT

The present invention provides efficient, economical, and environmentally-friendly compositions and methods for removing pollutants from water sources. In particular embodiments, the present invention provides compositions and methods for catalyzing the degradation of pharmaceutical pollutants in wastewater using the enzyme chloroperoxidase (CPO). Another embodiment provides a method of degrading pollutants in wastewater and other water sources. In specific embodiments, the claimed composition and method can be used to degrade pharmaceutical pollutants selected from the group consisting of: acetaminophen, carbamazepine, sulfamethazine, diclofenac, and naproxen.

CROSS-REFERENCE TO RELATE APPLICATION

This application is a continuation application of U.S. application Ser.No. 15/609,452, filed May 31, 2017, which is incorporated herein byreference in its entirety, including any figures, tables and drawing.

GOVERNMENT SUPPORT

This invention was made with government support under grant numberCHE0540763, awarded by the National Science Foundation. The governmenthas certain rights in the invention.

BACKGROUND OF INVENTION

Water systems are routinely monitored for the presence of pollutants,such as bacteria, viruses, pesticides, petroleum, acids, metals, andother chemicals. Increasingly, in recent years, pollution fromprescription drugs and over-the-counter (OTC) medications has causedconcern among water quality experts and environmental advocates.Pharmaceutical compounds have been detected in lakes, rivers, andstreams, in amounts ranging from nanograms per liter to micrograms perliter. Even in small concentrations exposure to pharmaceuticalpollutants has been shown to cause harm to aquatic ecosystems, such as,for example, altered sex ratios in certain fish populations. While therisk of low concentrations of pharmaceuticals in water appears to beminor for humans, sound information about the true effects and risks ofchronic drug exposure due to pharmaceutical water pollution is lacking.

Pharmaceutical compounds that enter aquatic environments mainly comefrom human and veterinary medicines, such as antibiotics,antidepressants, blood thinners, hormones, and painkillers. Sometimes,unused or expired medications enter waterways as a result of improperdisposal, for example, by flushing them down the toilet or drain.Pharmaceuticals can also enter water systems as a result of human andanimal drug consumption. The body metabolizes only a fraction of mostingested drugs. The remainder is excreted through sweat, urine, orfeces, which in turn ends up in wastewater systems. In the case ofantibiotic- or hormone-fed livestock in large-scale feeding operations,these unmetabolized drug compounds can leach into groundwater from thetons of regularly produced manure. Furthermore, some medications areapplied as creams or lotions rather than being ingested. The portions ofthese medications that are not absorbed into the skin are washed offinto wastewater as well.

While there have been certain efforts to prevent improper disposal ofpharmaceuticals, such as drug take-back programs and disposal guidelinesfrom the federal Environmental Protection Agency (EPA), these efforts donot effectively capture all drugs that might be disposed of in watersystems. Thus, wastewater treatment is an important measure to helpremove those remaining pharmaceutical pollutants that do end up in thewater.

There are multiple types of wastewater treatment. Activated sludgeinvolves the use of air, bacteria, and/or protozoa to degrade organicmaterials and remove nutrients from sewage and industrial waste waters.However, the efficiency of the activated sludge process in removingpollutants varies based on, for example, the temperature of degradationand hydraulic retention time for the various drug compounds.Additionally, activated sludge systems consume large amounts of energy.Also, construction, maintenance, and operation can be costly.

Other conventional systems include biological filtration, which is oftenutilized for municipal wastewater. These methods are insufficient toeliminate all persistent pharmaceutical residues, however, because ofthe diversity of drug properties. Additionally, sewage treatment resultsin the production of semi-solid waste byproducts, which require furthertreatment before being suitable for disposal.

Advanced processes such as UV, ozonation, microfiltration, andultrasound can achieve degradation of drug compounds close to, or at100%, efficiency. Nevertheless, these methods are inconsistent as wellbecause of the complexity of pharmaceutical pollutants.

Given the increase in pharmaceutical and personal care product use, itis unclear whether these existing efforts will be sufficient to combatpharmaceutical water pollution, due to limitations such as cost,efficiency, secondary byproducts, and inconsistency.

Thus, further investigations into the removal and/or breakdown of widelyused drugs in the environment, as well as improved methods of treatingdrug contamination in water sources, are warranted to prevent potentiallong-term consequences to the health of humans, other living organisms,and the environment.

BRIEF SUMMARY

The present invention provides efficient, economical, andenvironmentally-friendly compositions and methods for removingpollutants from water sources. In particular embodiments, the presentinvention provides compositions and methods for degrading pharmaceuticalpollutants in wastewater using the enzyme chloroperoxidase (CPO).

One embodiment of the subject invention provides a composition for thetreatment of wastewater and other water sources comprising the enzymechloroperoxidase, an oxidant, and a halogen ion. In one embodiment, theoxidant is hydrogen peroxide. In a further embodiment, the halogen ionis chloride or bromide.

In some embodiments, chloroperoxidase is in its pure form. The growthmedium of Caldariomyces fumago functions similarly to the pure form ofCPO. In other embodiments, chloroperoxidase is used in a more crudeprotein form.

Advantageously, the compositions of the subject invention can be used atlow concentrations to degrade pharmaceutical pollutants in water. Inspecific embodiments, the pharmaceutical pollutants can be one or moredrugs from the following classes: acetaminophen, carbamazepine,sulfamethazine, diclofenac, naproxen, or any other drug sharing similarstructures with these compounds.

Another embodiment provides methods for degrading pollutants inwastewater or other polluted water sources comprising administering aneffective amount of the composition to the wastewater or water source;allowing the composition to catalyze the degradation of pollutantswithin the wastewater or water source; and allowing the pollutants tobecome sufficiently degraded.

In one embodiment, the method of the subject invention can be used todegrade pharmaceutical pollutants.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the crystal structure of CPO, as represented by Pymolsoftware (protein database (PDB) entry 1CPO). The heme prophryin, is thelattice-like structure located in the center of the protein.

FIG. 2 shows a slice of CPO crystal structure with surfacerepresentation. The heme is the lighter-shaded structure depicted at thecenter. The narrow and wide channels are marked by arrows. Bromide isrepresented by lighter shaded spheres, adjacent to numbers 1 and 2.Iodide is represented by the darker spheres adjacent to numbers 1through 3. The halide binding sites are labeled by numbers 1 through 3.

FIG. 3 shows the active site of CPO with important amino acids in thedistal pocket, as represented by Pymol software (PDB entry 1CPO). Theheme is depicted at the center. His 105 was proposed to form hydrogenbonds with Glu 183 and Asp 106, thus conferring the proper position ofGlu183 for forming Compound I. Phe 103 and Phe 186 were observed inclose proximity to the heme center, suggesting their control oversubstrate access to the heme center by polarity.

FIG. 4 shows the oxidation of2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), catalyzedby CPO.

FIG. 5 shows the CPO-catalyzed dehalogenation of trihalophenols.

FIG. 6 shows the mechanism of CPO-catalyzed one-electron oxidation offluorophenol.

FIGS. 7A-7C show different examples of CPO-catalyzed reactions inphosphate buffer with chloride ions; 7A shows chlorination ofmonochlorodimedone (MCD); 7B shows halogenation by 1,3-cyclopentanedioneand alkenes; 7C shows exemplary epoxidation and hydroxylation reactions,including yields and enantiomeric purities.

FIG. 8 shows CPO-catalyzed chlorination of aromatic substrates inphosphate buffer with chloride ions.

FIG. 9 shows the general mechanism of a CPO-catalyzed reaction. AHrepresents the substrate. Compounds I and II represent the ferrylintermediates. X represents halogen atoms (except F⁻).

FIG. 10 shows a graphical representation of the UV-Visible (UV-Vis)spectra of CPO-catalyzed degradation ofN-acetyl-p-aminophenol/Acetaminophen (APAP), APAP with H₂O₂, and APAPmetabolites at 1 min, 2.5 min, and 4 min.

FIG. 11 shows a table listing Accurate-Mass LC-Q-TOF-MS data foridentification of APAP and its metabolites.

FIG. 12 shows the targeted MS/MS spectra of APAP and its metabolites.

FIG. 13 shows a graph depicting UV-Vis spectra of CPO-catalyzeddegradation of carbamazepine (CBZ) with its metabolites and H₂O₂ at 1min., 2.5 min., and 4 min.

FIG. 14 shows the effect of chloride on the degradation of CBZ.

FIG. 15 shows the effect of pH on CBZ degradation.

FIG. 16 shows the effect of H₂O₂ concentration on degradation rates.

FIG. 17 shows the effect of CBZ concentration on degradation rates.

FIG. 18 shows a table listing kinetic parameters of CPO-catalyzeddegradation of CBZ.

FIG. 19 shows the targeted MS/MS spectra of CBZ and its metabolites.

FIG. 20 shows a graph depicting the area of CBZ metabolites with limitedH₂O₂ at 1 min, 3 min, and 5 min detected by Accurate-Mass LC-Q-TOF-MS.

FIG. 21 shows the chromatograms of CBZ and its metabolites.

FIG. 22 shows the optimizer data of CM4, CM5, and CM7. The bold valueswere the mass of precursor ions. The italic values were estimated as thesame fragments adducted with varying hydrogens.

FIG. 23 shows the proposed mechanism of CPO-catalyzed degradation ofCBZ.

FIG. 24 shows a graph depicting the UV-Vis spectra of CPO-catalyzeddegradation of sulfamethazine (SMZ), with its metabolites and H₂O₂ at 1min. and 2.5 min.

FIG. 25 shows a table listing the Accurate-Mass QTOF-LC/MS data foridentification of SMZ and its metabolites catalyzed in a CPO—H₂O₂—Cl⁻system.

FIG. 26 shows a graphical depiction of the area of SMZ metabolites withlimited H₂O₂ at 1 min, 3 min, and 5 min detected by Accurate-MassLC-Q-TOF-MS.

FIG. 27 shows the proposed mechanism of the degradation of SMZ catalyzedby a CPO—H₂O₂—Cl⁻ system, represented by the primary structure of eachmetabolite.

FIG. 28 shows Accurate-Mass LC-Q-TOF-MS data for identification of SMZand its metabolites catalyzed by a CPO—H₂O₂—Br⁻ system.

FIG. 29 shows the mechanism of CPO-catalyzed bromination of SMZ.

FIG. 30A shows the chemical structure of diclofenac.

FIG. 30B shows the chemical structure of naproxen.

FIGS. 31A-31D generally show the relationship between degradationefficiency and reaction condition of CPO-catalyzed degradation ofdiclofenac (left) and naproxen (right).

FIG. 32 shows the proposed degradation pathway of diclofenac duringCPO-catalytic oxidative process.

FIG. 33A shows proton nuclear magnetic resonance (NMR) nuclearoverhauser enhancement spectroscopy (NOESY) of naproxen product [I]. Theweak nuclear overhauser effects (NOEs) between peaks a and c and betweenpeaks b and c can only be observed at lower contour levels. The NOEcoupling patterns unequivocally define the structure of this degradationproduct.

FIG. 33B shows proton NMR correlation spectroscopy (COSY) of naproxenproduct [I].

FIG. 34 shows COSY of naproxen.

FIG. 35 shows NOESY of naproxen.

FIG. 36 shows the proposed degradation pathway of naproxen during theCPO-catalytic oxidative process.

FIG. 37 shows a table listing elimination of COD and TOC byCPO-catalyzed oxidative degradation.

FIG. 38 shows COSY of authentic desmethylnaproxen purchased from SigmaAldrich.

FIGS. 39A, 39B, and 39C show levels of cell viability with regard toincreasing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) concentration for APAP, CMZ, and SMZ, respectively, comparedwith their metabolites.

DETAILED DISCLOSURE

The present invention provides efficient, economical, andenvironmentally-friendly compositions and methods for treatingwastewater polluted by pharmaceuticals. In particular embodiments, thepresent invention provides compositions and methods for degradingpharmaceutical pollutants in wastewater using the enzymechloroperoxidase (CPO).

One embodiment of the subject invention provides a composition fortreating wastewater or another source of water comprisingremedially-effective amounts of each of: the enzyme chloroperoxidase, anoxidant, and a halogen ion, wherein the remedially-effective amounts arecombined to form a system capable of degrading drug pollutants presentin the wastewater or water source.

Chloroperoxidase Enzyme

Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by thefungus Caldariomyces fumago. FIG. 1 shows the crystal structure of CPO,with its heme porphyrin depicted in red at the center of the protein.

FIG. 2 shows a slice of the crystal structure of CPO. As depicted byarrows, there is a narrow channel connecting the protein surface to theheme center. Halogen ions, such as bromide and iodide, bind with sitesin the narrow channel, suggesting that this channel provides the pathwayfor halides to reach the heme active center from the protein surface. Inaddition, the surface contains another, wider channel. This channel islikely the site where bulky molecules bind, and where reactions such asoxidation and epoxidation occur. Advantageously, CPO catalyzes two majortypes of oxidation: one-electron oxidations, similar to mostperoxidases, and two-electron oxidations, which are rare forconventional peroxidases.

FIG. 3 shows a representation of the CPO active site. The heme activesite of CPO resembles that of P450s and peroxidase. In conventional hemeperoxidases, histidine residue acts as an acid-base catalyst. CPOdiffers in that it utilizes glutamic acid 108 (Glu 183).

Generally, in reactions catalyzed by heme peroxidases, the distalacid-base catalyst forms hydrogen bond networks with other amino acidsto achieve the cleavage of the O—O bond of hydrogen peroxide. In CPO,Glu 183 reacts with histidine 105 (His 105). His 105 forms hydrogenbonds with Glu 183 and Asp 106, facilitating cleavage of the peroxidebond and proper positioning of Glu 183 in relation to the heme center.Two phenylalanine residues (Phe 103 and Phe 186) are in close proximityto the heme iron, and are thought to interact with hydrophobicsubstrates to control access to the heme center.

CPO-Catalyzed Reactions

The ability of CPO to catalyze one-electron oxidations, and two-electronoxidations, facilitates the use of CPO in the treatment of environmentalpollution.

CPO-catalyzed one-electron oxidations include peroxidation, anddehalogenation/polymerization driven by radicals.

FIG. 4 shows the oxidation of2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) catalyzed byCPO. This reaction typifies CPO-catalyzed peroxidation, and is used asan ABTS assay to measure the peroxidase activity of CPO and/or mutantstrains of CPO.

Additionally, dehalogenation has been found in trihalophenols andfluorophenols. FIG. 5 shows dehalogenation of trihalophenols catalyzedby CPO.

FIG. 6 shows the mechanism of CPO-catalyzed one-electron oxidation offluorophenols. According to this general mechanism, fluoride is notinvolved in CPO-catalyzed halogenation but is involved indehalogenation. The function of polymerization shown by some otherperoxidases is also found in CPO-catalyzed reactions. In thedefluorination of fluorophenols, for example, dimers and trimers aregenerated.

The two-electron oxidations catalyzed by CPO include halogenation andoxygen transfer. The reaction depicted in FIG. 7A shows CPO catalyzedchlorination of monochlorodimedone (MCD). This reaction is routinelyused as an MCD assay to measure the chlorination activity of CPO and/ormutant strains of CPO. Halogenation can also be carried out by bromideor iodide ions.

FIG. 8 shows that halogenation of other substrates can be catalyzed byCPO, such as phenols and, as FIG. 7B shows, 1,3-cyclopentanedione andalkenes. The oxidative chlorination catalyzed by CPO for halogenatedphenols or phenols typically generates different isomers. FIG. 7C showsother important two-electron oxidations, including epoxidation withenantioselectivity and hydroxylation.

Mechanism of CPO-Catalyzed Reactions

FIG. 9 shows the general catalytic cycle of CPO, starting from theresting state of the enzyme. The native ferric center binds with H₂O₂ atthe heme iron to form an oxo-ferryl cation radical intermediate,Compound I. During the reaction, two electrons from the heme center aretransferred to H₂O₂, cleaving the O—O bond and producing H₂O.

Compound I is involved in multiple reaction pathways. In the“dismutation” or “catalase” pathway, Compound I continues to react withH₂O₂ to generate O₂ and H₂O and is reduced back to its resting state. Inthe “P450” pathway, an organic substrate (RH) is hydroxylated andCompound I is reduced back to its ferric resting state.

In the “peroxidation” pathway, another reactive oxo-ferryl intermediate,Compound II, is formed. One electron from Compound I is transferred toan organic molecule (AH), converting AH to a radical molecule (A.). Theelectron is transferred to the organic substrate from Compound II, andCPO is returned to its resting state.

In the “halogenation” pathway, Compound I interacts with a halide (X) toform Compound X, a ferric intermediate. Compound X will halogenate anorganic substrate (AH) and generate a hydroxyl ion (OH⁻). It thenreturns back to its resting state.

Compound I, with its strong oxidizing capabilities, also generateshypochlorous acid and/or hypobromous acid as active intermediates inoxidative chlorination.

Selected Definitions

As used herein, a “remedially-effective amount” of the composition orany component of the composition is any amount or concentration thatwill result in the treatment or remediation of polluted or contaminatedwastewater or other water sources.

As used herein, “treatment” or “remediation” of wastewater or otherwater sources refers to the act or process of, or the result of,correcting a fault or deficiency, such as in modulating, ameliorating,reducing, reversing, or stopping damage or harm to the health of humans,other organisms, or the environment. This is achieved by acting uponcontaminants in the environment, such as hazardous or pollutingmaterials, which may be changed chemically, or physically, or degraded,stabilized or sequestered, or in some other way removed from thesurrounding environment. Treatment may comprise degradation and/orreduction of pollutants in water and can include the use of thecompositions of the present invention, either alone or in combinationwith other known treatment methods, including but not limited to UVtreatment, activated sludge, and bioremediation.

As used herein, “degradation” of a chemical compound can be usedinterchangeably with “dissolution,” “decomposition” and “digestion,” andrefers to the breakdown or separation of the compound into two or moresimpler products. The products resulting from degradation of a chemicalcompound can be, for example, simpler compounds, metabolites, orelemental parts.

As used herein, “bioremediation,” means using biological organisms,alone or in conjunction with inert structures, as a system for treating,modulating or altering the contaminants, such as hazardous or pollutingmaterials.

As used interchangeably herein, “contaminant” or “pollutant” means anymolecule, chemical or organism in the environment that is harmful toother living organisms in the environment or to the abiotic elements ofthe environment, and includes compounds or molecules that are in anamount greater than is desired for that environment even if suchcompounds or molecules are not inherently harmful if found in loweramounts. Biological, chemical, physical, or radiological substances(normally absent in the environment) which, in sufficient concentration,can adversely affect living organisms through air, water, soil, and/orfood are included in the terms “contaminant” or “pollutant.” The term,“toxic materials” as used herein, is included in the term “contaminant.”Pollutants may also be a natural element of the environment that ispresent in such a concentration that it is now harmful to theenvironment and its constituents. The pollutant may be an element thathas been introduced into the environment by human or animal activities,such as synthesis of the material, or by natural causes.

Contaminant or pollutant, as used herein, also means any molecules,chemicals or organisms in the environment that are present in anundesired concentration or amount. The contaminant may not necessarilybe harming any component of the environment but may be present in anundesired quantity.

Contaminant or pollutant, as used herein, also encompasses the phrases“pharmaceutical pollutant” and “environmentally persistentpharmaceutical pollutant (EPPP),” which refer to any compoundspecifically designed and/or produced for use by humans or animals as amedicinal drug or for personal care, which is obtained either byprescription or OTC, and which has entered, persisted, and/ordisseminated in the environment. Non-limiting examples of pharmaceuticalpollutants include compounds from the following classes of drugs:acetaminophen, amoxicillin, azithromycin, bacitracin, ciprofloxacinhydrochloride, doxycycline, erythromycin, lincomycin, naproxen,penicillin G, penicillin V, sulfadiazine, sulfamethazine,sulfamethizole, sulfamethoxazole, tetracycline, trimethoprim,diclofenac, carbamazepine, atenolol, bezafibrate, lidocaine,clarithromycin, diatrizoate, iopamidol, iopromide, cyclophosphamide, andifosfamide.

The term, “environment” as used herein, is defined generally as thesite, surroundings or conditions in which a person, animal, or plantlives or operates, and more specifically in terms of remediation, as anarea as defined by the contaminant/pollutant situation. Environment mayinclude the biotic and abiotic elements, and the patterns of theinterrelationships between the biotic elements, and between the bioticand abiotic elements that are found in the defined area. All threephysical states (solids, liquids, and gases) may be included in theelements that make up the environment.

The term environment also encompasses the phrases “wastewater” and“water source.” Wastewater refers to any water, the quality of which hasbeen adversely affected by human or animal activities. Such activitiescan include domestic, industrial, commercial, or agriculturalactivities. Further included in the term wastewater are surface runoff,or storm water, and municipal wastewater, or sewage. A water source isany body of water in the environment, either naturally occurring orman-made, including groundwater, aquifers, rivers, streams, lakes,ponds, and the like. Water sources can include but are not limited towater used for drinking, recreation, or habitation.

As used herein, the terms “isolated,” and “purified,” refer to materialthat is substantially or essentially free from components that normallyaccompany the compound as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques. Particularly, in preferred embodiments, the compound is atleast 85% pure, more preferably at least 90% pure, more preferably atleast 95% pure, and most preferably at least 99% pure.

Compositions of the Subject Invention

One embodiment of the subject invention provides a composition fortreating wastewater or another source of water comprisingremedially-effective amounts of the enzyme chloroperoxidase, an oxidant,and a halogen ion, wherein the remedially-effective amounts are combinedto form a system capable of degrading pollutants present in thewastewater or water source. Any reference to a “CPO—H₂O₂—X⁻ system”,where X is a halide, is intended as a reference to a compositionaccording to the subject invention.

In preferred embodiments, the concentration of hydrogen peroxide andCPO, as well as the pH level of the composition of the subjectinvention, are optimized based on the actual substrate being treatedwith the composition.

In some embodiments, chloroperoxidase is in its pure form. The growthmedium of Caldariomyces fumago functions similarly to the pure form ofCPO. In other embodiments, chloroperoxidase is used in a crude proteinform.

In some embodiments, isolated CPO is present in the composition in aremedially-effective amount. CPO concentration preferably ranges fromabout 0.1 nM to about 50 nM. In a specific embodiment, when, e.g.,diclofenac is the substrate being treated, CPO is present at a rangefrom about 0.25 nM to about 6.0 nM, preferably at a concentration above5.0 nM. In another embodiment, when, e.g., naproxen is the substratebeing treated, CPO is present at a range from about 1.0 to about 23.0nM, preferably at a concentration above 20.0 nM. In yet anotherembodiment, when, e.g., APAP, CBZ, or SMZ is the substrate beingtreated, CPO is present at a range from about 0.4 nM to about 5.0 nM.

In another embodiment, the oxidant of the claimed composition is ahydroperoxide. In specific embodiments, the oxidant is hydrogen peroxide(H₂O₂). Preferably, hydrogen peroxide is present in the composition inan amount below that which causes degradation of CPO. It is well knownthat high concentration of H₂O₂ (an oxidant) inactivates most hemecontaining enzymes due to internal oxidative destruction of theporphyrin prosthetic group. Thus, low concentrations of H₂O₂ areemployed for reactions catalyzed by most heme peroxidases. However, thisstrategy cannot be simply applied to CPO because of its ability tocatalyze the disproportionation (i.e., a redox reaction producing twodifferent products from the oxidation and reduction of the same element)of hydrogen peroxide. Therefore, optimum concentration of hydrogenperoxide depends on the actual substrate being degraded, and includesthe amount of substrate that is being degraded.

In specific embodiments of the present invention, hydrogen peroxide ispresent in the composition at a concentration from about 0.03 mM toabout 2.0 mM, more preferably from about 0.1 mM to about 0.7 mM, andeven more preferably from about 0.3 mM to about 0.5 mM.

Hydrogen peroxide concentration can also be determined with respect tothe amount of substrate being treated. For example, in certainembodiments, the ratio of concentration of substrate to hydrogenperoxide is 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

In another embodiment, the composition further comprises aremedially-effective amount of a halogen ion. The halogen ion can beselected from chloride, bromide, and iodide. In another embodiment, thehalogen ion is obtained by the addition of a remedially-effective amountof halide salt, such as potassium chloride or potassium bromide. Thehalogen ion can be present at, for example, a concentration ranging fromabout 0.01 mM to about 50 mM, preferably from about 0.05 to about 30 mM,more preferably from about 5.0 mM to about 25 mM, even more preferablyfrom about 15 mM to about 20 mM.

In another embodiment, the CPO is conjugated to poly[hydroxyethylmethacrylate-co-(poly(ethylene glycol)-methacrylate] membranes tostabilize the CPO and increase the recyclability of the enzyme. This canenhance the storage and thermal stability of CPO versus that of freeCPO.

Use of the Composition for Water Treatment

The composition of the subject invention can be used to catalyzedegradation of pollutants in wastewater or other water sources. Inspecific embodiments, the pollutant is a pharmaceutical pollutant.

In particular embodiments, the pharmaceutical pollutant is from one ofthe following classes of drugs: acetaminophen, carbamazepine,sulfamethazine, diclofenac, and naproxen. These pharmaceuticals arecommonly used in human and veterinary medicine and persist in theenvironment.

Another embodiment provides methods for treating wastewater or otherpolluted water sources comprising administering a remedially-effectiveamount of the composition to the wastewater or water source; allowingthe composition to catalyze degradation of pollutants within the watersource; and allowing the water pollutants to sufficiently degrade.

In a particular embodiment, sufficient degradation is achieved whentreatment or remediation of the water has occurred. In anotherembodiment, sufficient degradation can be total degradation of waterpollutants.

The rate of degradation can vary depending on certain factors, such asthe drug compound being treated and the concentration of oxidant and/orCPO applied. In certain embodiments, sufficient degradation occurs inless than 1 minute. In other embodiments, the sufficient degradationtakes from about 1 minute up to about 5 minutes, to about 10 minutes, toabout 1 hour, to about 24 hours, or longer.

The claimed method can be carried out at relatively low pH. In specificembodiments, the present method is carried out within a pH range ofabout 2.0 to about 5.0, preferably within the range of about 2.5 toabout 3.5, and even more preferably within the range of about 2.75 toabout 3.2.

In certain embodiments, the method can be carried out in combinationwith other wastewater treatment methods. Metabolites and/or products ofdrug compounds can be susceptible to removal via biological treatments,activated sludge, or UV treatment, for example. Thus, products can beremoved using these secondary treatment processes, providing evengreater degradation efficiency for water pollutants.

In another embodiment, exemplified below, methods are provided foridentifying the metabolites of acetaminophen (APAP), carbamazepine(CBZ), and/or sulfamethazine (SMZ). Another embodiment provides methodsfor identifying the products of CPO-catalyzed degradation of diclofenacand naproxen. Further embodiments provide methods for determining thedegradation pathways of APAP, CBZ, SMZ, diclofenac, and naproxen.

In another embodiment, the method further comprises the step of testingthe wastewater or water source in which the composition of the subjectinvention is administered for the presence of known drug metabolitesand/or products after degradation has occurred.

The examples described below illustrate exemplary embodiments of thematerials and methods of the subject invention. These exemplaryembodiments should not be construed as limiting the scope of the subjectinvention.

Example 1—CPO-Catalyzed Chlorination and Polymerization of AcetaminophenAcetaminophen

Acetaminophen (N-acetyl-p-aminophenol, APAP), also known as paracetamol,is an active ingredient in many OTC medications. It is widely used as apain reliever and fever reducer.

The following experiments provide investigations into the efficiency ofCPO-catalyzed degradation of APAP, while also proposing the degradationpathway and metabolites of APAP. As noted previously, CPO contains ananalogous proximal heme iron thiolate ligand structure to that of P450,but for degradation of APAP, the CPO—H₂O₂—Cl⁻ system works differentlyfrom a P450-hypochlorite system. Mainly, chlorination and dimerizationare preferred over oxidation, suggesting CPO possesses specialcapabilities for detoxification of APAP.

Experimental Materials and Methods

The isolation of CPO proposed in this study was a modification of theprotocol reported by Morris and Hager (1966), using acetone instead ofethanol as the fractionation solvent. CPO with Rz=1.45 was applied inall reactions (Rz is the purity standard, defined as A398/A280=1.44 fora pure enzyme). All solvents were HPLC grade or Optima® LC/MS grade,purchased from Thermo Fisher Scientific Inc. (Waltham, Mass., USA).Water was produced using a Milli-Q® Biocel™ Ultra-Pure waterpurification system equipped with 0.22 μM membrane filter cartridge (EMDMillipore, Billerica, Mass., USA), and an organic removal cartridge(Evoqua Water Technologies, Lowell, Mass., USA).

UV-Visible Spectrophotometry

A VARIAN UV-Vis spectrophotometer (Cary 200 Bio) was used to collect theUV spectra of the degradation products. The drug solution was scanned bydissolving 0.11 mM APAP in 100 mM KH₂PO₄ buffer with 20 mM KCl at pH2.75. The same solution was monitored after being mixed with 0.55 mMH₂O₂. The reaction was initiated with the addition of 5 nM CPO and theUV-Vis spectrum was recorded for reaction times of 1 min., 2.5 min., and4 min.

Liquid Chromatography and Mass Spectrometry

To investigate the degradation efficiency, 62.56 μM APAP was reactedwith 321.56 μM H₂O₂ and 0.43 nM CPO for 1 hour at room temperature. Themixture was centrifuged at 3,000 g in Centriprep® centrifugal filterunit with a 30,000 Dalton cut-off membrane (EMD Millipore, Billerica,Mass., USA). The yielded product was collected after being centrifugedfor 1 min. Ethyl acetate was used to extract the filtrate while shakingvigorously, and the supernatant was evaporated to dryness using nitrogengas. The dried metabolites were dissolved in H₂O/methanol (95:5 v/v) toachieve a final concentration of 1 mg/L (ppm). Each sample was eitherstored in a freezer at −20° C. or immediately run in the LC-Q-TOF-MSmass spectrometer system.

A low concentration of CPO and H₂O₂ reaction sample was prepared asfollows: 6.86 μM H₂O₂ was added directly to Centriprep® centrifugalfilter unit with the same membrane size as described above. Then, 66.20μM APAP was added, catalyzed by the addition of 1.28 nM CPO in 100 mMphosphate buffer with 20 mM KCl for 3 min. and 5 min. The experimentswere run in triplicate. Dried metabolites were dissolved in H₂O/methanol(95:5 v/v) to achieve a concentration of ˜1 mg/L (ppm) and analyzed bythe LC-Q-TOF-MS system immediately.

Next, a high concentration of APAP sample was prepared by mixing 413 μMAPAP with 2 mM H₂O₂, catalyzed by 2 nM CPO for 35 min. at roomtemperature. The extraction was carried out by ethyl acetate, and theorganic layer was dried using nitrogen gas. Metabolites were dissolvedin 1.5 mL H₂O/methanol (95:5 v/v) to achieve an approximate finalconcentration of 5 mg/L (ppm). The sample was filtrated through a 0.22μM polyethersulfone syringe filter. The sample was stored in a freezerat −20° C. and analyzed using both a Q-TOF-LC-MS and Triple-quadrupoleLC-MS/MS system.

Instrumentation and Chromatographic Separation

Chromatographic separation and identification of metabolites wasperformed using the Agilent 1290 Infinity UPLC system coupled withAgilent 6530 Q-TOF mass spectrometer. The Agilent MassHunter DataAcquisition Software (rev. B0.06.00) was used to control the UPLC andthe mass spectrometer. Mass Hunter Qualitative Data Analysis Software(Rev. B.07.00) was used for data mining and identification. An AgilentZORBAX Eclipse plus C18 Rapid Resolution High Definition (RRHD) column(100 mm×1.8 μm) was applied, with the column temperature set at 30° C.via a thermostatted column compartment (TCC). An infinity 1290 automaticinjector was used to inject 1 μL of the sample to the column. A flowrate of 0.4 mL/min was used with an aqueous mobile phase A, consistingof water with 5 mM ammonium formate and 0.1% formic acid, and an organicmobile phase B, consisting of acetonitrile with 0.1% formic acid. Theoptimized chromatographic gradient was at 0 minutes, 95% A, and 5% B. Bincreased linearly to 95% over 8 min. and this gradient was maintainedfor 1 min. The positive ionization mode was applied during allexperiments.

In full scan MS mode, the accurate mass data of the molecular ions wereprocessed through the Agilent MassHunter Qualitative Analysis software.The collected retention times and confirmed formulas of every metabolitewere applied in targeted MS/MS mode to identify metabolite information.

Additionally, an Agilent 6460 Triple-Quadrupole LC/MS/MS massspectrometer was used to increase the detection sensitivity of theisolated metabolites that were initially identified by the LC-QTOF massspectrometer. The same column and elution program previously used in theLC-QTOF was employed in the LC-QQQ-MS system.

Results UV-Vis Study of CPO-Catalyzed Degradation of APAP

The UV-Vis spectra of APAP (0.11 mM) were scanned in 100 mM KH₂PO₄buffer with 20 mM KCl at pH 2.75. FIG. 10 shows the spectra of APAP withH₂O₂ and its metabolites. APAP showed a strong absorption at 242 nm.After mixing with 0.55 mM H₂O₂, the absorption curve increased slightlydue to absorbance of H₂O₂. One minute after addition of 5 nM CPO, the477 nm absorption decreased, then increased at 4 minutes Absorption at218 nm increased after 1 minute, and stopped increasing at 2.5 minutes.

The same reaction was carried out under the same conditions, but withoutthe chloride ion (KCl) in the phosphate buffer. Without KCl, theabsorbance did not change after the addition of CPO, meaning the Cl⁻ isnecessary in the degradation of APAP in the CPO—H₂O₂—Cl⁻ system.

Degradation Efficiency—LC-Q-TOF-MS

62.56 μM APAP was reacted with H₂O₂ (321.56 μM) and CPO (0.43 nM) for 1hour. The sample was analyzed by LC-Q-TOF-MS. APAP was not observed,meaning degradation efficiency of 100% was achieved.

Seven (7) APAP metabolites (coded AM1 to AM7) were confirmed bydifferent retention times and accurate mass-to-charge ratios (m/z). FIG.11 shows the elemental formula, retention time, relative mass differencebetween observed mass and mass of the target compound (ppm), anddifference between the observed mass and mass of the target compound (inmilliDaltons) for each metabolite.

The structures of each metabolite were confirmed in targeted MS/MS byretention time and fragment ions. FIG. 12 shows the targeted MS/MSspectra of APAP and its metabolites.

Discussion

When APAP mixes with hypochlorite, toxic products such as1,4-benzoquinone and N-acetyl-p-benzoquinone imine (NAPQI) have beenreported. These products were not detected in the CPO—H₂O₂—Cl⁻ system.Rather, evaluation of the toxicity of the final products indicated thatCPO resulted in excellent purification of the wastewater when comparedwith commonly used chlorine disinfection systems.

Example 2—CPO-Catalyzed Degradation of Carbamazepine

Carbamazepine (CBZ) is widely used in the treatment of epilepsy,trigeminal neuralgia and bipolar disorder. CBZ is detectable in surfacewater, ground water, and even drinking water. Various harmful ecologicaleffects, for example in fish and rodents, suggest the possibility thatlong term exposure to CBZ in drinking water is also a potential risk tohuman health.

Degradation efficiency in water, kinetic parameters, degradationpathways, and the products of CPO-catalyzed degradation of CBZ wereinvestigated in this experiment under optimized conditions, includingconcentrations of C1- and H₂O₂, and pH.

UV-Visible Spectrophotometry

A VARIAN UV-Vis spectrophotometer (Cary 200 Bio) was used to collect theUV spectra of the degradation products. The drug solution was scanned bydissolving 0.07 mM CBZ in 100 mM KH₂PO₄ buffer with 20 mM KCl at pH2.75. The same solution was monitored after being mixed with 0.35 mMH₂O₂. UV spectra were recorded after the addition of 5 nM CPO for 1min., 2.5 min., and 4 min.

The effect of the concentration of chloride was investigated.Degradation was carried out in 100 mM phosphate buffer at pH 3.0 with0.07 mM CBZ and 0.07 mM H₂O₂. Concentration of chloride was increasedfrom 0 to 20 mM. 285 nm wavelength was monitored immediately after 2.5nM CPO was added.

The degradation rate at pH range of 2.0 to 5.0 was investigated to probethe effect of pH on the CPO-catalyzed reaction. The reaction was carriedout in 100 mM phosphate buffer with 20 mM KCl, 0.07 mM CBZ, and 0.07 mMH₂O₂. H₃PO₄ and KOH were used to adjust pH. 285 nm wavelength wasmonitored immediately after the addition of 2.5 nM CPO. The calculatedtime was 0.2 to 0.4 min.

The degradation rate of CBZ was determined by different concentrationsof H₂O₂, ranging from 0.07 to 0.71 mM, in 100 mM phosphate buffer with20 mM KCl and 0.07 mM CBZ at pH 3.0. A wavelength of 285 nm wasmonitored immediately after the addition of 5 nM CPO. The degradationrate was calculated at 0.1 to 0.3 min.

The rate was calculated by monitoring absorbance at 285 nm from 0.1 to0.2 min. The reaction was carried out in 100 mM KH₂PO₄ buffer with 20 mMKCl at pH 2.75, with the concentration of CBZ varying from 0.02 to 0.11mM. UV monitoring was started immediately after adding 0.35 mM H₂O₂.

All experiments were triplicated and data reported were mean values ofthree independent measurements with standard deviation.

Liquid Chromatography and Mass Spectroscopy

To investigate degradation efficiency, CBZ was dissolved in methanol tomake stock solution (21.19 mM). 10.59 μM (2.5 mg/L) CBZ was reacted with107.19 μM H₂O₂ and 2.9 nM CPO for 10 min. at room temperature. Thesample was prepared using the same technique as described in Example 1.The sample was stored at −20° C. in a freezer, or immediately run in aLC-Q-TOF-MS mass spectrometer system.

To detect all CBZ metabolites, CBZ was dissolved in methanol to makestock solution (21.19 mM). A high concentration of CBZ sample wasprepared by mixing 62.56 μM (14.8 mg/L) CBZ with 0.4 nM CPO. H₂O₂ stocksolution (41.16 mM) was added to the reaction system at 56.5 μL/minuteto bring the final concentration of H₂O₂ up to 316.56 μM. The totalreaction time was 55 min. The solution was extracted by ethyl acetate,and the supernatant was evaporated by using nitrogen gas. Metaboliteswere dissolved in 2.0 mL H₂O/methanol (95:5 v/v) to achieve a finalconcentration of ˜5 mg/mL (ppm). The sample was centrifuged at 1200 gfor 10 min., and 1.5 mL of supernatant was removed by syringe.Filtration was applied using a 0.22 μM polyethersulfone syringe filter.The sample was stored in a freezer at −20° C., and analyzed both using aLC-Q-TOF/MS system and a Triple-Quadrupole LC/MS/MS system.

To investigate the mechanism of degradation, samples were prepared witha low H₂O₂ concentration. 6.86 μM H₂O₂ was added directly to Centriprep®centrifugal filter unit (30,000 Dalton), along with 42.28 μM CBZcatalyzed by 1.3 nM CPO in the 100 mM phosphate buffer with 20 mM KClfor 1 min., 3 min., and 5 min. Filter units were centrifuged at 3000 gfor 1 min. at room temperature. The filtrate was extracted by ethylacetate, and the organic phase was purged with nitrogen gas to dryness.The dried metabolites were dissolved in H₂O/methanol (95:5 v/v) to makethe final concentration approximately 1 mg/L (ppm), and immediatelyanalyzed using a LC-Q-TOF-MS system. Experiments were run in duplicate.

The instrumentation and chromatographic separation were the same as thatin Example 1.

Results UV-Vis Study of CPO-Catalyzed Degradation of CBZ

FIG. 13 shows UV-Vis spectra of CBZ with H₂O₂ and its metabolites at 1min., 2 min., and 4 min. The spectrum of CBZ showed a strong absorptionat 285 nm. 1 min. after addition of 5 nM CPO, the 285 nm absorptiondecreased, and stopped increasing at 4 min. The 285 nm wavelength wasused to measure kinetic parameters of CBZ degradation catalyzed by CPO.

The same reaction was carried out under the same conditions, except forthe chlorine ion (KCl) was absent in the phosphate buffer. Without KCl,the absorbance did not change after the addition of CPO. The chlorineion is necessary in the degradation of CBZ in the CPO—H₂O₂—Cl⁻ system.

The efficiency of degradation catalyzed by 5 nM CPO, calculated by thechange of absorbance at 285 nm, was 96% within 4 minutes; however, theefficiency might be more than 96% because the tail of the UV peak of theproduct appeared to cover the original drug (285 nm).

Effect of Chloride, pH and Hydrogen Peroxide Concentration on theDegradation of CBZ

FIG. 14 shows the effect of chloride on the degradation of CBZ. Thedegradation rate was calculated to range from 0.2 to 0.6 min. Thedegradation rate increased from 0 to 18.89 M/min. The observation wasconsistent with those described above, suggesting that chlorideparticipates in the degradation of CBZ.

FIG. 15 shows the effect of pH on CBZ degradation. The optimum pH forthe degradation of CBZ is 3.0 in the presence of 20 mM Cl⁻. Thedegradation efficiency decreased at a pH of 3.5 to 5. This suggestedthat CPO could be applied in an acid wastewater treatment.

FIG. 16 shows the effect of hydrogen peroxide concentration ondegradation. The degradation rate was increased as the concentration ofH₂O₂ increased to 0.53 mM. From 0.53 to 0.71 mM, the rate was stillstable, with only a slight decrease. This could be due to thedegradation of CPO by the high concentration of H₂O₂. (Manoj, 2010). Thestability of CPO in the presence of H₂O₂ compared with other peroxidasessuggests that CPO has greater prospects for application than otherswithin the same enzyme class.

FIG. 17 shows the effects of different concentrations of CBZ whenreacted with 0.07 mM H₂O₂ and 5 nM CPO. FIG. 18 shows the calculatedkinetic parameters of CBZ degradation catalyzed by CPO.

LC-Q-TOF-MS

To investigate the degradation efficiency of CBZ in the CPO—H₂O₂—Cl⁻system, CBZ was reacted with H₂O₂ and CPO for 10 minutes. The sample wasanalyzed by LC-Q-TOF-MS, and CBZ was not observed, showing that adegradation efficiency of 100% was achieved even by low concentrationsof CPO. This establishes the utility of CPO treatment in large-scalewastewater treatment.

FIG. 19 shows the targeted MS/MS spectra of CBZ and its metabolites. Thestructures of each metabolite were confirmed by retention time andfragment ions. There were two types of metabolites, distinguished basedon their structure. The first type contained CM1, CM2, CM9, CM10, andsuggested oxygen insertion into the CBZ-based structure. The other typeof structure was based on acridine (CM5), the decarbonylation product ofthe parent compound. All chlorinated metabolites were fromacridine-based structures.

To investigate the degradation mechanism, this experiment was carriedout for a limited reaction time (1, 3, and 5 min.) with lowconcentration of H₂O₂ (6.86 μM). FIG. 20 shows the area of metabolitesdetected from the reaction. The ratio of the concentrations of CBZ andH₂O₂ was about 7:1. Only part of the CBZ was degraded, and only somemetabolites were detected, which were potentially the first metabolitesformed.

At the first minute, only CM1, CM2, CM9, and CM10 were formed. The fourproposed structures were suggestive of oxygen insertion into theCBZ-based structure, produced directly from CBZ without decarbonylation.CM1 was the most abundant metabolite, suggesting that the first step ofdegradation was the epoxidation of CBZ. The percentage of these fourcompounds decreased significantly after being reacted with higherconcentration of H₂O₂ for longer periods of time. At 3 min., acridine(CM5) was formed. CM7 and CM8, two acridine-based metabolites, weredetected at 5 min., with final concentrations at relatively high levels.

Compared with the CBZ-based compounds, acridine-based structuresappeared to comprise the major products. Acridine (CM5), thedecarbonylation product of the parent compound, with CM6, CM7, and CM8were the most abundant metabolites of the CPO-catalyzed degradation,while CBZ-based structures existed as intermediates of the degradation.

The chlorinated products, CM11-CM15, were observed in relatively lowconcentrations (together comprising 1.96% of total metabolites). Thesemetabolites were not observed at the first stage of the reaction. Thus,these products were not major products of the CPO-catalyzed degradationreaction.

FIG. 21 shows the chromatograms of CBZ and its metabolites with theirproposed structures. In the chromatograms of CM1, CM2, CM9, CM11, CM12,CM13, CM14, and CM15, isomers were identified by their differentretention times. CM1, CM9, CM11, and CM12 had problems with signal tonoise ratio, which might be due to low concentrations.

FIG. 22 shows the proposed mechanism of CPO-catalyzed metabolism of CBZ.

Discussion

CPO-catalyzed degradation of CBZ was efficient, using nanomolar-levelCPO concentrations. CBZ was depleted by 100% using CPO concentration of2.5 to 14.8 mg/L for 10 min., or ≥96% with 16.5 mg/L for 4 min. Thedegradation rate and concentrations were conservative: removal abilityis mostly likely greater than the parameters of the experiment, as theLC samples were not monitored by shorter time periods.

Compared with some biological treatment in water, such as white rotfungus, the efficiency of degradation was dramatically improved by CPOcatalyzation. Strong degradation ability with high concentrations of CBZusing simple conditions (low concentration of catalyst, easy operation,and proper reaction time) show the potential for the composition andmethod for wastewater treatment.

Example 3—CPO-Catalyzed Degradation of Sulfamethazine

Sulfonamide pharmaceutical substances are widely used in human andveterinary antibacterial treatments. These substances are frequentlydetected in wastewater and surface water.

Sulfamethazine (SMZ), or sulfadimidine, belongs to a group ofheterocyclic sulfonamides. It has been detected in livestock manure andin surface water. Additionally, SMZ-resistant bacteria have been foundin water samples, implying that extensive SMZ use has increased the riskof antibacterial resistance and could be detrimental to human health.

To evaluate the CPO—H₂O₂—Cl⁻ system for degradation of antibiotics, thereaction efficiency of CPO in the degradation of SMZ was investigated,and the degradation pathway and structures of metabolites were proposed.

UV-Visible Spectrophotometry

A VARIAN UV-Vis spectrophotometer (Cary 200 Bio) was used to collect theUV spectra of the degradation products. SMZ was dissolved in methanol tomake a 3.59 mM stock solution. The drug solution was scanned bydissolving 0.06 mM SMZ in a 100 mM KH₂PO₄ buffer with 20 mM KCl at pH2.75. It was then mixed with 0.06 mM H₂O₂. UV spectra were recordedafter the addition of 5 nM CPO for 1 min., 2.5 min., and 4 min.

Liquid Chromatography and Mass Spectrometry

To investigate the degradation efficiency, SMZ was dissolved in methanolto make a stock solution (3.59 mM). 62.45 μM (17.38 mg) of SMZ was mixedwith 1.3 nM CPO for 30 min. at room temperature. H₂O₂ stock solution(41.16 mM) was added to reaction system at 56.5 μL/min. to achieve afinal concentration of 314 M H₂O₂. The sample was prepared using thesame technique described in Example 2. The sample was stored in afreezer at −20° C., or immediately analyzed using a LC-Q-TOF-MS massspectrometer system.

To detect all metabolites of SMZ, the same experiment above was carriedout for 1.5 hours. The filtrate was extracted directly by ethyl acetate,and nitrogen gas purge was used to dry the sample. The dried metaboliteswere dissolved in 2.0 mL H₂O/methanol (95:5 v/v) to produce aconcentration of approximately 5 mg/L (ppm). The sample was centrifugedwith 1200 g for 10 min. and then 1.5 mL of supernatant were removed bysyringe. Filtration was performed using a 0.22 M polyethersulfonesyringe filter. The sample was stored in a freezer at −20° C. andanalyzed using a LC-Q-TOF-MS system.

To investigate the mechanism of degradation in the CPO—H₂O₂—Cl⁻ system,the sample was prepared with low concentration of H₂O₂. 6.86 μM H₂O₂ wasadded directly to Centriprep® centrifugal filter unit (30,000 Daltoncut-off) with 35.90 μM SMZ, catalyzed by 1.3 nM CPO in the 100 mMphosphate buffer with 20 mM KCl, for 1 min., 3 min., and 5 min. Filterunits were centrifuged at 3000 g for 1 min. at room temperature, and thefiltrate was extracted by ethyl acetate. The organic phase was purgedwith nitrogen gas to dryness. The dried metabolites were dissolved inH₂O/methanol (95:5 v/v) to produce a final concentration ofapproximately 1 mg/L (ppm), and detected by LC-Q-TOF-MS systemimmediately. Experiments ran in triplicate.

To investigate brominated products, the reaction was also carried out ina buffer containing KBr instead of KCl, along with 62.45 μM SMZ, 2 mMH₂O₂, and catalyzed by 2 nM CPO for 30 min. After the extraction andpurge procedure, the final concentration dissolved in the H₂O/methanol(95:5 v/v) was ˜1 mg/L (ppm).

Instrumentation and chromatographic separation were the same as inExample 1, but without the application of Triple-Quandrupole LC/MS/MSmass spectrometry.

Results UV-Vis Study of CPO-Catalyzed Degradation of Sulfamethazine

The spectra of SMZ showed absorption at 241 nm, 263 nm, and 306 nm. 1min. after addition of 5 nM CPO, the 241 nm absorption increased, andstopped increasing at 2.5 min., while the 263 nm peak kept decreasing.The wavelength at 306 nm increased from 1 min. to 2.5 min. The UVspectrum at 4 min. was monitored, and was the same as the wavelength at2.5 min. H₂O₂ was added to the mixture solution, to increase the totalconcentration of H₂O₂ to 0.30 mM. After 1 min., the 241 nm peakdecreased and a strong absorption was observed at 273 nm (FIG. 24).There was no wavelength that could be used to measure the kineticparameters of SMZ degradation catalyzed by CPO.

The same reaction was carried out under the same conditions except forthe absence of a chlorine ion (KCl) in the phosphate buffer. WithoutKCl, the absorbance did not change after the addition of CPO. Thechlorine ion is imperative in the degradation of SMZ in the CPO—H₂O₂—Cl⁻system.

CPO—H₂O₂—Cl⁻ System

The degradation efficiency of SMZ in CPO—H₂O₂—Cl⁻ system wasinvestigated. The sample was analyzed by LC-Q-TOF-MS and SMZ was notobserved. Thus, the degradation efficiency of 100% by nanomolar levelsof CPO was achieved, suggesting the potential for application of CPO inlarge-scale wastewater treatment.

LC-Q-TOF-MS

Samples were detected in Agilent Technologies 6530 Accurate-MassLC-Q-TOF-MS in full scan MS mode, the accurate mass data of themolecular ions were processed through the Agilent MassHunter QualitativeAnalysis software. To detect all metabolites, samples were concentratedby five-fold. There were 8 SMZ metabolites (coded SM1 to SM8), confirmedby different retention times and accurate mass-to-charge ratios (m/z).SM9 was detected only in the reaction with limited H₂O₂ within 5 min. asan intermediate product. FIG. 25 shows the elemental formula, retentiontime, relative mass difference between the observed mass and the mass ofthe target compound (in ppm), and the difference between the observedmass and the mass of target compound (in milliDaltons), as collected.

The relative mass differences of the standard drug (SMZ) and metaboliteswere less than 4.0 ppm. The mass differences of all compounds were lessthan 1.0 mDa. This method was proved to be efficient for determining themetabolites of SMZ.

As shown in FIG. 26, to investigate the mechanism of the degradationprocess, the experiment was carried out for a limited reaction time (1,3, and 5 minutes) with low concentration of H₂O₂ (6.86 μM). The ratio ofdrug concentration to H₂O₂ concentration was about 5:1.

FIG. 27 shows the proposed pathway for SMZ metabolism catalyzed by CPO,with each metabolite shown by one of its proposed chemical structures.For the CPO—H₂O₂—Cl⁻ system, the major pathways were the chlorinatedsteps with one and two chlorine atom addition and desulfonylation ofSMZ. The minor pathway was hydroxylation and chlorination with threechlorine atoms.

CPO—H₂O₂—Br⁻ System

To investigate the halogenation of SMZ catalyzed by CPO, 20 mM KBr wasapplied in the buffer instead of KCl. The CPO—H₂O₂—Br⁻ system reactionwas carried out at room temperature, and the product spectrum wasobtained. As shown in FIG. 28, there were only 3 metabolites detected,all of which were brominated products. The ratios of the moleculescontaining three bromine atoms were 1:3:3:1. As shown in FIG. 29, whichdepicts the mechanism of SMZ bromination catalyzed by CPO, there wasonly one pathway to elucidate the mechanism of SMZ halogenation.

Discussion

The process of CPO-catalyzed SMZ degradation was efficient. 100%depletion of SMZ was achieved by a nanomolar level of CPO. Thedegradation rate and substrate concentrations were conservative: removalcapability was mostly likely greater than the parameters used in theexperiment because LC samples were not monitored by a shorter timeperiod.

Compared with some biodegradation treatment in water, such as white rotfungus, the efficiency of CPO-catalyzed reaction was dramaticallyimproved. Given the nanomolar amount of enzyme required and the rapidsample preparation, CPO-catalyzed degradation can be used in large-scalewastewater treatment.

Example 4—Degradation of Non-Steroidal Anti-Inflammatory DrugsDiclofenac and Naproxen by CPO

FIG. 30A shows the chemical structure of diclofenac, and FIG. 30B showsthe chemical structure of naproxen. Diclofenac(2-[(2,6-dichlorophenyl)amino]benzeneacetic acid; sodium salt) andnaproxen (2-(6-methoxynaphthalen-2-yl)propionic acid) are non-steroidalanti-inflammatory drugs (NSAIDs), widely used for the treatment ofarthritis, ankylosing spondylitis, and acute muscle pain. However, mostNSAIDs are usually not completely metabolized by the human body, andsimply pass through. Furthermore, these compounds are difficult to breakdown using general waste treatment strategies.

The following is a study of the degradation of diclofenac and naproxenby CPO. The major degradation products were identified and reactionpathways were postulated. Results demonstrated that CPO can effectivelyconvert both diclofenac and naproxen into compounds that aresignificantly less toxic based on their inhibitory effects and EC50value on the growth of a freshwater green alga, Chlorella pyrenoidos.

Experimental Materials and Methods

CPO was isolated from the growth medium of C. fumago according to themethod of Morris and Hager (1966) with minor modifications, usingacetone rather than ethanol in the solvent fractionation step. CPO withRz=1.03 (A398/A280, 1.44 for pure enzyme) was prepared and stored in 100mM phosphate buffer (pH 5.0) at 4° C.

The halogenation activity of CPO used in this study was 4232 U·mL⁻¹based on the standard MCD assay (Hager et al., 1966). The aromatichydroxylation activity (3563 U·mL-1) of the enzyme was determined bymonitoring the hydroxylation of naphthalene into I-naphthol (Kluge etal., 2007). The classic peroxidase activity of the enzyme determinedusing ABTS as the substrate was 3071 U·mL⁻¹ (Manoj et al., 2008).

All reagents used in this study, including diclofenac, naproxen,dipotassium hydrogen phosphate, potassium dihydrogen phosphate, hydrogenperoxide (30% in aqueous solution), ethyl acetate, and inorganicreagents for cultivating the green alga were obtained from Xi'anChemical Co. Ltd (Xi'an, China) with highest purity (≥98%). Otherchemicals, such as methanol and acetonitrile (chromatography grade), aswell as standard degradation products of the two drugs,o-desmethylnaproxen and 4′-hydroxydiclofenac (chromatography grade),were purchased from Sigma Aldrich (St. Louis, Mo. USA).

Phosphate buffer (0.1 mol·L⁻¹) was prepared by mixing appropriatevolumes of 1 mol·L⁻¹ KH₂PO₄ and K₂HPO₄ stock solutions and diluting thecombined solutions to 1 L. The solution was adjusted to various pH with1 mol·L⁻¹ H₃PO₄. All solutions were prepared using deionized water witha conductivity of 5.6×10-8 s·cm-1.

Degradation of Diclofenac and Naproxen

Enzymatic degradation of both drugs was carried out in 0.1 mol·L-1phosphate buffer in a centrifugal tube with a total volume of 3.0 mLcontaining CPO (0.25-0.23 nmol·L-1), 20 mmol·L-1 KCl, and drugs (15μmol·L⁻¹) at pH 2˜6 at 298K. The reaction was started by adding H₂O₂(0.015-0.3 mmol·L⁻¹) in the absence of light under magnetic stirring andwas continued for 20 min. The supernatant of the reaction mixture wasextracted 3 times using ethyl acetate. The combined organic extract wasconcentrated by rotary evaporation (0.09 MPa, 308K) to remove thesolvent. The extracts were then dissolved in acetonitrile and methanol,respectively, for HPLC (LC-20AT, Shimadzu) analysis. The mobile phaseconsisted of 80:20 (v/v) acetonitrile and water for diclofenac and 90:10(v/v) methanol and water for naproxen, and the flow rate was 0.5ml·min⁻¹. The detector was set at 274 nm and 235 nm for diclofenac andnaproxen, respectively. The quantitative analysis of the targetcompounds was based on the standard curve (correlation coefficientswere >0.999). The effect of reaction parameters (pH, concentration ofenzyme/H₂O₂, and reaction time) on degradation efficiency wasinvestigated and optimized.

All experiments were triplicated and data reported were mean values ofthree independent measurements.

Determination of Products

Samples were treated as above for HPLC-MS analysis. An Esquire LC-iontrap mass spectrometer (Bruker Daltonics, Germany) equipped with anorthogonal geometry Electrospray Ionization (ESI) source was employed todetermine the formulae of the products. Nitrogen was used as the drying(8 L·min⁻¹) and nebulizing (0.8 bar) gas at 180° C. Scanning wasperformed from m/z 100 to 1000 in the standard resolution mode.

To establish the structure of the degradation products, the reaction wascarried out using the same condition as stated above except a largervolume (300 mL) was used. Upon completion of the reaction, the reactionmixture was extracted with either ethyl acetate or chloroform. Afterremoval of the solvent by rotary evaporation (0.09 MPa, 308K), theextracts were dissolved in either deuterated chloroform or methanol andtransferred to 5 mm NMR tubes. NMR experiments were carried out on aBruker 600 MHz NMR spectrometer operating at a proton frequency of599.73 MHz. All spectra were recorded at 298 K using standard pulseprograms from the manufacturer.

Elimination of COD and TOC

Total organic carbon (TOC) measurement was conducted on a TOC-VCPAanalyzer (Shimadzu Corp.). The feed speed was 150 ml·min⁻¹. Chemicaloxygen demand (COD) was measured by a quick method on a RapidAutoanalyzer (5B-1(F), Lian-hua Tech. Co., Ltd). A solution containing2.5 ml sample, 0.7 ml reagent D (potassium dichromate) and 4.8 mlreagent E (catalysts) in a 20.0 ml glass tube was heated to 438K andkept for 10 min. together with a blank sample and a standard sample.After 2 min. of air cooling, the heated solution was cooled by water foranother 2 min. Then the absorbency of the samples was measured at 610nm.

Treatment of Drug Effluent by Activated Sludge

A mixed population of activated sludge microorganisms was collected fromXi'an second sewage treatment plant (Xi'an, China). The sample ofactivated sludge was filtered on a Spectra Mesh polypropylene 149-μmfilter (Spectrum Laboratories Inc., Rancho Dominguez, Calif., USA) toremove aggregates. The sample was then washed three times bycentrifugation and suspended in the same volume of culture medium. Toremove any excessive amounts of dissolved organic carbon, the suspensionwas stirred and maintained under oxygen at 298K for at least 24 hwithout exposure to the test materials. The volatile ratio f ofactivated sludge was 0.74.

90 mL of 15 μmol·L⁻¹ drug effluent was put into the activated sludgesuspension before/after enzymatic treatment (to ensure a final drugconcentration of 7.5 μmol·L⁻¹). The samples were stirred for theduration of the study with a magnetically coupled stirrer when air wasused as the aerated gas. The sample was then left to stand for 1-2 h.The supernatant was taken for determination of COD.

Toxicity Tests

Freshwater unicellular green alga, C. pyrenoidosa (provided by theInstitute of Wuhan Hydrobiology, Chinese Academic of Science), wascultivated in nutrient media of blue green medium (BG11) at 298K andilluminated with cool-white fluorescent lights at a continuous lightintensity of 2000 Lx. For cell experiments, C. pyrenoidosa was exposed,during its log growth phase, to the toxicant at five differentconcentrations (maintain final concentration ranging from 0.01 to 0.36mg/L) for 3-4 days at 298K. The concentration of the alga was determinedby monitoring the change of absorption at 680 nm (Ma et al., 2006). Thetoxicity tests for each drug concentration were conducted in triplicate.

The growth inhibition rate for each sample was calculated. EC50 (drugconcentration required to cause 50% reduction in growth) values werecalculated using linear regression analysis of drug concentration asnatural logarithm versus percentage inhibition. All correlationcoefficients were >0.99.

Results Effect of Reaction Parameters on Drug Degradation

Since various physicochemical parameters influence the degradationefficiency, it is essential to optimize these factors in order to makethe process more efficient and practically applicable.

FIGS. 31A-31D generally show the relationship between degradationefficiency and reaction condition of CPO-catalyzed degradation ofdiclofenac (left) and naproxen (right).

The pH range investigated was 2-6 due to poor stability and activity ofCPO at higher pH. FIG. 31A shows that the degradation efficiency ofnaproxen and diclofenac increased rapidly with increasing pH, andreached maximum around pH 3.2. The degradation efficiency decreasedsharply when pH was increased above 3.2.

The effect of H₂O₂ concentration on CPO-catalyzed degradation ofdiclofenac and naproxen is shown in FIG. 31B. As expected for allperoxidases, no degradation of either drug was observed before theaddition of H₂O₂. The degradation efficiency of both drugs increased asH₂O₂ concentration is increased. Maximum rate is achieved when totalH₂O₂ concentration reached 0.1 mmol·L⁻¹. Further addition of H₂O₂,however, repressed the degradation of both drugs possibly due toformation of Compound III caused by high concentrations of H₂O₂ (Ayalaet al., 2011). Therefore, 0.1 mmol·L⁻¹ of H₂O₂ was chosen in thedegradation of both diclofenac and naproxen in all subsequentexperiments.

Optimizing CPO concentration not only improves degradation efficiencybut also saves cost of operation. The range of CPO concentration testedwas 0.25-6.0 nmol·L⁻¹ for diclofenac and 1.0-23.0 nmol·L⁻¹ for naproxen.FIG. 31C shows that degradation efficiency of both drugs increasedrapidly as CPO concentration was increased. Complete degradation of thedrugs was achieved when CPO concentration was above 5.0 nmol·L⁻¹ fordiclofenac, and 20.0 nmol·L⁻¹ for naproxen, indicating that CPO isextremely efficient in the degradation of the subject drugs. CPOdegrades both diclofenac and naproxen with a remarkable rate. As shownin FIG. 31D, about 70% of diclofenac and 75% of naproxen is degradedwithin 1 min. The complete degradation was achieved in only 9 and 7 min.for diclofenac and naproxen, respectively, at optimum reactioncondition.

Determination of Products by HPLC-MS and NMR

HPLC-MS and NMR analyses were employed to establish the structures ofthe putative products from the two drugs. CPO-catalyzed hydroxylation ofdiclofenac and O-demethylation of naproxen were observed.

FIG. 32 shows the sequence of diclofenac degradation catalyzed by CPObased on the products identified from HPLC-MS and NMR analysis. Thediverse catalytic activity of CPO makes it possible to produce a broadarray of products from diclofenac degradation, however, under theconditions employed in this study, only hydroxylation activity wasobserved. This can be appreciated by the similarity between CPO andcytochrome P450 that metabolizes most xenobiotics via hydroxylation.Thus CPO converts diclofenac to either monohydroxylated ordihydroxylated products, the same as the major products observed in themetabolism of diclofenac (Blum et al., 1996; Osorio et al., 2014).

The identification of products from CPO-catalyzed degradation ofnaproxen was achieved by detailed NMR analysis of the products with theaid of MS. FIGS. 33A and 33B show proton NMR NOESY spectra and COSYspectra of naproxen product [I], respectively. The most noticeabledifference between proton NMR spectra of the products and the parentdrug, naproxen, is the absence of the methoxy signal (peak “i” around3.9 ppm, as shown in FIGS. 34 and 35) in the products. This suggests thedemethylation of naproxen.

Although CPO catalyzed N-demethylation has been reported (Kedderis etal., 1980), the observed O-demethylation represents novel CPO activity.This is reminiscent of the activity displayed by P450 (Meunier et al.,2004) and UPOs (Hofrichter et al., 2014; Kinne et al., 2009) that arestructurally related to CPO.

Based on the major products identified from that study, the reactionsequence of naproxen degradation catalyzed by CPO was proposed, as shownin FIG. 36. Similar to the degradation of diclofenac, CPO catalyzeddegradation of naproxen is achieved primarily via CPO's monooxygenaseactivity. Thus, CPO degrades naproxen to either monohydroxylated ordihydroxylated products, similar to the major products observed inbacterial degradation of naproxen (Wojcieszyńiska et al., 2014).Initially, naproxen was degraded to desmethylnaproxen (Aresta et al.,2006; Urrea et al., 2010).

The products identified in the above NMR and MS analysis are subjectedto further oxidative transformation as supported by the results fromactivated sludge experiment as well as eco-toxicity test.

Reduction in Chemical Oxygen Demand (COD) and Total Organic Carbon (TOC)

FIG. 37 showed that only 4.9%, 9.1% of COD and 25%, 7.6% of TOC removalwas achieved for diclofenac and naproxen, respectively. It is thereforeproposed that CPO catalyzed degradation can serve as an efficientpre-treatment step in waste water treatment. This can be combined withsubsequent bioremediation technologies (activated sludge) for completedecontamination of the two drugs in waste water.

Combined Treatment of Drug Effluent by Enzymatic Oxidation and ActivatedSludge

As indicated in FIG. 37, the COD value of drug effluent did not decreasenoticeably after CPO-treatment. However, compared with the parent drugs,the products from CPO catalyzed degradation have improved solubility inaqueous media and are more vulnerable to further biodegradation. Thisconclusion was confirmed by the observation that treatment ofCPO-catalyzed reaction mixture with activated sludge increased CODremoval from 4.9% and 9.1% to 85% and 86% for diclofenac and naproxen,respectively. On the other hand, treatment by activated sludge aloneonly removes 49% and 54%, of the COD for diclofenac and naproxen,respectively, suggesting that more effective decontamination of the twodrugs can be achieved through CPO pre-treatment followed by existingbioremediation technologies (activated sludge).

Evaluation of the Eco-Toxicity of the Products

In some cases simple destruction of a drug is inadequate, since theresulting products may also be highly toxic, and special attention musttherefore be paid to toxicity assessment of products to ensure that theagent has been effectively detoxified. However, toxicity evaluationabout the products from diclofenac and naproxen are not readilyavailable. Biological assays offer a direct measure to evaluate themagnitude of the potential health risk of chemicals. Therefore, agrowth-inhibitory test was carried out using C. Pyrenoidosa.

FIG. 38 shows COSY of authentic desmethylnaproxen purchased from SigmaAldrich. This showed that the 72-h EC₅₀ increased with the increase indegradation efficiency. The value was 0.25 mg·L⁻¹ for diclofenac and0.33 mg·L⁻¹ for naproxen at the end of degradation. These resultsdemonstrated that the products had lower toxicity compared with theparent drugs, suggesting the great potential of using CPO as anefficient catalyst in the safe removal of these drugs fromenvironmental.

Discussion

This study demonstrated that CPO catalyzed oxidative degradation is apromising alternative for treatment of waste water containingnon-steroidal anti-inflammatory drugs. Complete degradation ofdiclofenac and naproxen is reached in only 9 and 7 min, respectively,under mild conditions.

The products identified by HPLC-MS suggested the initial hydroxylationof the drug molecules followed by further oxidative transformation. Thebiodegradability of the decomposition products was significantlyincreased as confirmed by COD measurement after combining the enzymaticoxidation with activated sludge treatment. Most significantly, theproducts of both diclofenac and naproxen had dramatically lower toxicitythan the original drugs as judged by the 72-h EC₅₀ value of C.Pyrenoidos. Our results demonstrate that CPO can serve as an efficient,cost-effective, and environmentally friendly catalyst for large-scaletreatment of waste water contaminated with the two drugs studied.

Example 5—MTT Assays of Acetaminophen, Carbamazepine, and SulfamethazineExperimental Materials and Methods

Cells (MDA-MB-231 Breast Cancer Cells; 2×105) were plated in 6-wellplates in DMEM containing 10% FBS. Cells were incubated in the absenceor in the presence of different amounts (0 to 2 μg/ml) of differentcompounds as showed in the figure for 24 h.

Cells were exposed to 10 mg/ml MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] for thelast 4 h. After incubation, the medium was removed and formazan crystalswere dissolved in detergent reagent following the manufacturer'sinstructions (ATCC). Optical density for each condition was determinedat 570 nm.

MTT incorporation was expressed as percentage of the control in theabsence of compounds. DMSO (0.04%) and methanol (0.001%) were used ascontrol since these compounds were dissolved on them at 10 mg/mL. Theconcentration is expressed in μg/ml as percentage of control (100%methanol was added at 0.001%). FIGS. 39A, 39B, and 39C show levels ofcell viability for each drug with respect to increasing MTTconcentration for ACT and ACT metabolites.

Results

Acetaminophen (ACT) MTT (μg/ml) ACT ACT-Metabolites ACT-SD ACT_met-SD 0100% 100% 4.00% 8% 0.0425  70%  90% 6.00% 7% 0.85  50%  80% 5.00% 4% 1.7 10%  80% 5.00% 6% Carbamazepine (CBZ) MTT (μg/ml) CBZ CBZ-MetabolitesCBZ-SD CBZ_met-SD 0 100% 100%   5% 7% 0.0425  80%  90%   8% 5% 0.85  60% 80%   3% 2% 1.7  45%  70%   5% 9% Sutfamethazine (SMZ) MTT (μg/ml) SMZSMZ-Metabolites SMZ-SD SMZ_met-SD 0 100% 100%   6% 8% 0.0425  80%  90%  4% 4% 0.85  50%  95%   8% 6% 1.7  40%  90%   2% 1%

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1. A composition for degrading pharmaceutical pollutants in wastewater,or another source of water, said composition comprisingremedially-effective amounts of chloroperoxidase, an oxidant, a halogenion and a phosphate buffer, said remedially-effective amount ofchloroperoxidase comprising about 1.0 nM to about 50 nMchloroperoxidase, and said composition having a pH value of about 2.5 toabout 3.5.
 2. The composition of claim 1, wherein the chloroperoxidasehas a purity value of Rz=1.45.
 3. The composition of claim 1, whereinthe chloroperoxidase comprises growth medium of Caldariomyces fumago. 4.(canceled)
 5. The composition of claim 1, wherein theremedially-effective amount of chloroperoxidase is about 5.0 nM to about6.0 nM.
 6. The composition of claim 1, wherein the remedially-effectiveamount of chloroperoxidase is about 20.0 nM to about 23.0 nM.
 7. Thecomposition of claim 1, wherein the remedially-effective amount ofchloroperoxidase is about 1.0 nM to about 5.0 nM.
 8. The composition ofclaim 1, wherein the oxidant is hydrogen peroxide.
 9. (canceled)
 10. Thecomposition of claim 1, said composition comprising the oxidant at aconcentration of about 0.03 mM to about 2.0 mM.
 11. The composition ofclaim 1, wherein the halogen ion is chloride or bromide.
 12. Thecomposition of claim 11, wherein the halogen ion is chloride.
 13. Thecomposition of claim 1, wherein the halogen ion is in the form of ahalide salt.
 14. The composition of claim 13, wherein the halide salt ispotassium chloride or potassium bromide.
 15. The composition of claim 1,said composition comprising the halogen ion at a concentration of about5.0 mM to about 25 mM.
 16. The composition of claim 15, said compositioncomprising the halogen ion at a concentration of about 20 mM.
 17. Acomposition for treating wastewater, or another water source, saidcomposition comprising: chloroperoxidase at a concentration of about 0.1nM to about 20.0 nM; hydrogen peroxide at a concentration of about 0.03mM to about 2.0 mM; a chloride ion at a concentration of about 5.0 mM toabout 25 mM; and a phosphate buffer; said chloroperoxidase, hydrogenperoxide, and halogen ion combining to form a system capable ofdegrading one or more pollutants selected from acetaminophen,carbamazepine, sulfamethazine, diclofenac and naproxen present in thewastewater or other water source; and said composition having a pH valueof about 2.5 to about 3.5.
 18. The composition of claim 17, saidcomposition comprising chloroperoxidase at a concentration of about 0.5nM to about 6.0 nM.
 19. A composition for treating wastewater, oranother water source, said composition comprising: chloroperoxidase at aconcentration of about 20.0 nM to about 50.0 nM; hydrogen peroxide at aconcentration of about 0.3 mM to about 0.5 mM; a chloride ion at aconcentration of about 5.0 mM to about 25 mM; and a phosphate buffer;said chloroperoxidase, hydrogen peroxide, and halogen ion combining toform a system capable of degrading one or more pollutants selected fromacetaminophen, carbamazepine, sulfamethazine, diclofenac and naproxenpresent in the wastewater or other water source; and said compositionhaving a pH value of about 2.5 to about 3.5.
 20. The composition ofclaim 19, said composition comprising chloroperoxidase at aconcentration of about 20 nM to about 23 nM.